Our long range goal is to determine the mechanism of action of insect cation pumps and to define their role in epithelial function during development. Our present objectives are (1) to confirm a cellular K-transport route by electrical methods (2) to determine apparent kinetic constants (Km, Vmax, and KI) for the transport of Li, Na, K, Rb, and Cs (3) to study the route and kinetics of Mg transport (4) to study the relationship between the alkali metal pump and the magnesium pump (5) to isolate and purify proteins involved in potassium transport and (6) to reconstruct a K-transport system by adding purified transport proteins to a synthetic membrane system. The standard method will be to isolate the midgut as a flat sheet and to short-circuit it automatically when appropriate. The electrical methods will consist of probing the isolated midgut using a potassium sensitive microelectrode. The kinetic methods will consist of measuring unidirectional fluxes and extracellular spaces with isotopes and measuring ion concentrations with flame spectrophotometry. Calculations will be made by computer and constants will be expressed in terms of extracellular ion concentrations and in the case of K in terms of cellular activities as well. Transport proteins will be located from the kinetic analysis and by perturbing the isolated midgut with enzymes. Loose proteins will be released into the bathing medium by osmotic shock and bound proteins will be recovered from homogenates using conventional membrane fractionation procedures. They will be purified by slabs in the presence of gel electrophoresis on polyacrylamide sodium dodecyl sulfate using preparations from spinning larvae, which have lost the K-pump, as controls. Test membranes will consist of simple lipid lack monolayers or liposomes. The effects of adding purified components will be measured by changes in electrical potential differences, electrical resistance, and isotopic fluxes and will be judged by the recovery of kinetic constants measured in the intact isolated midgut.